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Lys48‐linked ubiquitin chains, regulating proteasomal protein degradation, are known to include cyclized forms. This cyclization hinders recognition by many downstream proteins by occluding the Ile44‐centered patch. In contrast, the A20‐like Znf domain of ZNF216 (a ubiquitin‐binding protein, A20 Znf) is expected to bind to cyclic ubiquitin chains via constitutively solvent‐exposed surfaces. However, the underlying interaction mechanism remains unclear. Here, our ITC and NMR experiments collectively showed that cyclization did not interfere with and even slightly enhance the molecular recognition of diubiquitin by A20 Znf. This effect is explained by the cyclization‐induced repression of conformational dynamics in diubiquitin and an enlarged molecular interface in the complex. Thus, these results suggest that cyclic ubiquitin chains can be involved in regulation of ZNF216‐dependent proteasomal protein degradation. [ABSTRACT FROM AUTHOR]

This article discusses the history and significance of autophagy, a cellular process that involves the degradation and recycling of intracellular material. The discovery of autophagy genes in yeast by Yoshinori Ohsumi 30 years ago paved the way for a surge of research in this field. The article highlights various aspects of autophagy, including its molecular mechanisms, regulation, and its role in human diseases such as cancer and neurodegeneration. The authors also raise important questions about the therapeutic potential of modulating autophagy and the future directions of autophagy research. [Extracted from the article]

Two related post‐translational modifications, the covalent linkage of Ubiquitin and the Small Ubiquitin‐related MOdifier (SUMO) to lysine residues, play key roles in the regulation of both DNA repair pathway choice and transcription. Whereas ubiquitination is generally associated with proteasome‐mediated protein degradation, the impact of sumoylation has been more mysterious. In the cell nucleus, sumoylation effects are largely mediated by the relocalization of the modified targets, particularly in response to DNA damage. This is governed in part by the concentration of SUMO protease at nuclear pores [Melchior, F et al. (2003) Trends Biochem Sci 28, 612–618; Ptak, C and Wozniak, RW (2017) Adv Exp Med Biol 963, 111–126]. We review here the roles of sumoylation in determining genomic locus positioning relative to the nuclear envelope and to nuclear pores, to facilitate repair and regulate transcription. [ABSTRACT FROM AUTHOR]

RNA‐binding proteins (RBPs) play vital roles in organisms through binding with RNAs to regulate their functions. Small molecules affecting the function of RBPs have been developed, providing new avenues for drug discovery. Herein, we describe the perspectives on developing small molecule regulators of RBPs. The following types of small molecule modulators are of great interest in drug discovery: small molecules binding to RBPs to affect interactions with RNA molecules, bifunctional molecules binding to RNA or RBP to influence their interactions, and other types of molecules that affect the stability of RNA or RBPs. Moreover, we emphasize that the bifunctional molecules may play important roles in small molecule development to overcome the challenges encountered in the process of drug discovery. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein 1 (BMP1), a member of the astacin family of zinc‐metalloproteases, proteolytically cleaves the low‐density lipoprotein receptor (LDLR) within its ligand‐binding domain, reducing the binding and cellular uptake of LDL‐cholesterol. Here, we aimed to determine whether astacin proteases other than BMP1 may also cleave LDLR. Although human hepatocytes express all six astacin proteases, including the meprins and mammalian tolloid, we found through pharmacological inhibition and genetic knockdown that only BMP1 contributed to the cleavage of LDLR in its ligand‐binding domain. We also found that the minimum amino acid change required to render mouse LDLR susceptible to cleavage by BMP1 is mutation at the P1′ and P2 positions of the cleavage site. When expressed in cells, the resulting humanised‐mouse LDLR internalised LDL‐cholesterol. This work provides insight into the biological mechanisms regulating LDLR function. [ABSTRACT FROM AUTHOR]

HUWE1, a HECT E3 ligase, is critical for processes like protein degradation and tumor development. Contrary to previous findings which suggested minimal non‐covalent interactions between the HUWE1 HECT domain and ubiquitin, we identified a non‐covalent interaction between the HUWE1 HECT N‐lobe and ubiquitin using NMR spectroscopy, revealing a conserved ubiquitin‐binding mode shared across HECT E3 ligases. Molecular dynamics simulations not only confirmed the stability of this interaction but also uncovered conformational changes in key residues, which likely influence binding affinity. Additionally, we highlighted the roles of both conserved and unique residues in ubiquitin binding. These findings advance our understanding of the interactions between the HUWE1 HECT domain and ubiquitin, and highlight potential targets for therapeutic intervention in the ubiquitin‐proteasome pathway. [ABSTRACT FROM AUTHOR]

Clear cell Renal Cell Carcinoma (ccRCC) is the most common and metastatic urological cancer. Molecular players of ccRCC progression and metastasis are not completely known. Here, using primary cell cultures from patients' specimens, we found that TGFβ1/Smad signalling is more activated in high versus low grade ccRCC and inversely correlates with Abl2 tyrosine kinase protein expression. TGFβ1 treatment increased ubiquitination and degradation of Abl2 protein in ccRCC cell lines by TGFβ1/Smad pathway activation and reactive oxygen species production. 3D invasion and matrix degradation assays showed that Abl2 promoted TGFβ1‐induced ccRCC cell invasion and maturation of invadopodia, a hallmark of tumour invasion and metastasis. Our findings define Abl2 as a new downstream molecule of TGFβ1 signalling and putative target to counteract advanced ccRCC. [ABSTRACT FROM AUTHOR]

Autophagy and Hippo signalling pathways both play important roles in cell homeostasis and are often involved in tumourigenesis. However, the crosstalk between these two signal pathways in response to stress conditions, such as nutrient deficiency, is incompletely understood. Here, we show that vesicular localised coiled‐coil domain containing 115 (CCDC115) inhibits autophagy as well as Hippo signalling pathway under starvation. Moreover, we show that CCDC115 interacts with the HOPS complex. This interaction competes with STX17, thus inhibiting the fusion of autophagosomes with lysosomes. Hence, CCDC115 inhibits the autophagic degradation of yes‐associated protein (YAP), thereby promoting cell proliferation in nutrient‐restricted situation. [ABSTRACT FROM AUTHOR]

The tumour suppressor p53 has been implicated in the host defence system against hepatitis C virus (HCV) infection, although the detailed mechanism remains unknown. Here, we found that p53 inhibits HCV replication by downregulating HCV Core protein levels in human hepatoma cells. For this effect, p53 potentiated the role of E6‐associated protein (E6AP) as an E3 ligase to induce ubiquitination and proteasomal degradation of HCV Core. Specifically, p53 facilitated the binding of E6AP to HCV Core through direct interactions with the two proteins. In addition, E6AP failed to induce ubiquitination of HCV Core in the absence of p53, suggesting that p53 increases the E3 ligase activity of E6AP in a triple complex consisting of p53, E6AP and HCV Core. [ABSTRACT FROM AUTHOR]

The conversion of zymogen Factor X (FX) to an active protease involves the removal of a 52-residue long activation peptide (AP). Through site-directed mutagenesis, we investigate the role of the AP and demonstrate that the high abundance of proline residues is important for efficient proteolysis of FX. Moreover, we identify an essential interaction site for Factor IXa (FIXa) between residues 22 and 30 (AP numbering) and find that the residues between 31 and 41 may provide an important interaction site for the intrinsic tenase complex, composed of Factor IXa (FIXa) and Factor VIIIa (FVIIIa). Finally, we suggest that the carbohydrate chain at Asn-39 restricts the activator specificity, as elimination of this glycosylation site increases the activation rate for activation by FIXa and FXa. [ABSTRACT FROM AUTHOR]

Linear ubiquitin chains play pivotal roles in immune signaling by augmenting NF-κB activation and suppressing programmed cell death induced by various stimuli. A20-binding inhibitor of NF-κB 1 (ABIN1) binds to linear ubiquitin chains and attenuates NF-κB activation and cell death induction. Although interactions with linear ubiquitin chains are thought to play a role in ABIN1- mediated suppression of NF-κB and cell death, the underlying molecular mechanisms remain unclear. Here, we show that upon stimulation by Toll-like receptor (TLR) ligands, ABIN1 is phosphorylated on Ser 83 and functions as a selective autophagy receptor. ABIN1 recognizes components of the MyD88 signaling complex via interaction with linear ubiquitin chains conjugated to components of the complex in TLR signaling, which leads to autophagic degradation of signaling proteins and attenuated NF-κB signaling. Our current findings indicate that phosphorylation and linear ubiquitination also play a role in downregulation of signaling via selective induction of autophagy. [ABSTRACT FROM AUTHOR]

Calmodulin is a conserved calcium signalling protein that regulates a wide range of cellular functions. Amino-terminal acetylation is a ubiquitous posttranslational modification that affects the majority of human proteins, to stabilise structure, as well as regulate function and proteolytic degradation. Here, we present data on the impact of amino-terminal acetylation upon structure and calcium signalling function of fission yeast calmodulin. We show that NatA-dependent acetylation stabilises the helical structure of the Schizosaccharomyces pombe calmodulin, impacting its ability to associate with myosin at endocytic foci. We go on to show that this conserved modification impacts both the calcium-binding capacity of yeast and human calmodulins. These findings have significant implications for research undertaken into this highly conserved essential protein. [ABSTRACT FROM AUTHOR]

The c‐Myc oncoprotein is frequently overexpressed in human cancers and is essential for cancer cell proliferation. The dysregulation of ubiquitin‐proteasome‐mediated degradation is one of the contributing factors to the upregulated expression of c‐Myc in human cancers. We herein identified USP17 as a novel deubiquitinating enzyme that regulates c‐Myc levels and controls cell proliferation and glycolysis. The overexpression of USP17 stabilized the c‐Myc protein by promoting its deubiquitination. In contrast, the knockdown of USP17 promoted c‐Myc degradation and reduced c‐Myc levels. The knockdown of USP17 also suppressed cell proliferation and glycolysis. Collectively, the present results reveal a novel role for USP17 in the regulation of c‐Myc stability and suggest its potential as a therapeutic target for cancer treatment. [ABSTRACT FROM AUTHOR]

Interleukin‐6 (IL‐6) and interleukin‐11 (IL‐11) are two important pleiotropic cytokines, both of which signal through a homodimer of the β‐receptor gp130. Specificity is gained through the unique, nonsignaling α‐receptors IL‐6R and IL‐11R. Soluble variants of IL‐6R and IL‐11R also exist. Both membrane‐bound receptors can be cleaved by the metalloprotease ADAM10. Here, we use ten different chimeric receptors consisting of different parts of IL‐6R and IL‐11R and analyze their susceptibility toward cleavage by ADAM10. As expected, all chimeras are substrates of ADAM10. However, we observed that cleavage of chimeric receptors containing the stalk region of the IL‐11R could be blocked by the protease inhibitor GI (selective for ADAM10), but not by the protease inhibitor GW (selective for both ADAM10 and ADAM17), suggesting that another protease besides ADAM10 is involved in cleavage of these chimeras. [ABSTRACT FROM AUTHOR]

In bacteria, Lon is a large hexameric ATP‐dependent protease that targets misfolded and also folded substrates, some of which are involved in cell division and survival of cellular stress. The N‐terminal domain of Lon facilitates substrate recognition, but how the domains confer such activity has remained unclear. Here, we report the full‐length structure of Lon protease from Thermus thermophilus at 3.9 Å resolution in a substrate‐engaged state. The six N‐terminal domains are arranged in three pairs, stabilized by coiled‐coil segments and forming an additional channel for substrate sensing and entry into the AAA+ ring. Sequence conservation analysis and proteolysis assays confirm that this architecture is required for the degradation of both folded and unfolded substrates in bacteria. [ABSTRACT FROM AUTHOR]

Maintenance of the proteome (proteostasis) is essential for cellular homeostasis and prevents cytotoxic stress responses that arise from protein misfolding. However, little is known about how different types of misfolded proteins impact homeostasis, especially when protein degradation pathways are compromised. We examined the effects of misfolded protein expression on yeast growth by characterizing a suite of substrates possessing the same aggregation‐prone domain but engaging different quality control pathways. We discovered that treatment with a proteasome inhibitor was more toxic in yeast expressing misfolded membrane proteins, and this growth defect was mirrored in yeast lacking a proteasome‐specific transcription factor, Rpn4p. These results highlight weaknesses in the proteostasis network's ability to handle the stress arising from an accumulation of misfolded membrane proteins. [ABSTRACT FROM AUTHOR]

Plant metacaspases type I (MCA‐Is), the closest structural homologs of caspases, are key proteases in stress‐induced regulated cell death processes in plants. However, no plant MCA‐Is have been characterized in vitro to date. Here, we show that only plant MCA‐Is contain a highly hydrophobic loop within the C terminus of their p10 domain. When removed, soluble and proteolytically active plant MCA‐Is can be designed and recombinantly produced. We show that the activity of MCA‐I depends on calcium ions and that removal of the hydrophobic loop does not affect cleavage and covalent binding to its inhibitor SERPIN. This novel approach will finally allow the development of tools to detect and manipulate the activity of these cysteine proteases in vivo and in planta. [ABSTRACT FROM AUTHOR]

Decoding of OAZ1 (Ornithine decarboxylase AntiZyme 1) mRNA, which harbours two open reading frames (ORF1 and ORF2) interrupted by a naturally occurring Premature Termination Codon (PTC), produces an 8 kDa truncated polypeptide termed Orf1p, unless the PTC is bypassed by +1 ribosomal frameshifting. In this study, we identified Orf1p as an endogenous ubiquitin‐dependent substrate of the 26S proteasome both in yeast and mammalian cells. Surprisingly, we found that the ribosome‐associated quality control factor Rqc1 and the ubiquitin ligase Ltn1 are critical for Orf1p degradation. In addition, the cytosolic protein quality control chaperone system Hsp70/Hsp90 and their corresponding co‐chaperones Sse1, Fes1, Sti1 and Cpr7 are also required for Orf1p proteolysis. Our study finds that Orf1p, which is naturally synthesized as a result of a premature translation termination event, requires the coordinated role of both ribosome‐associated and cytosolic protein quality control factors for its degradation. [ABSTRACT FROM AUTHOR]

Microtubule nucleation is mainly mediated by the γ‐tubulin ring complex (γTuRC), whose core components are γ‐tubulin and γ‐tubulin complex proteins GCP2–6. A substantial fraction of γ‐tubulin also exists with GCP2 and GCP3 in a tetramer called the γ‐tubulin small complex (γTuSC). To date, the mechanisms underlying the turnover of γ‐tubulin and GCPs have remained unclear. Here, we show that γ‐tubulin, GCP2, and GCP3 are proteolyzed by the ubiquitin‐proteasome system, and we identify cullin 1, cullin 4A, and cullin 4B as the E3 ligases that mediate the ubiquitination and, consequently, the degradation of γ‐tubulin. Notably, we found that γTuSC disassembly promotes the degradation of γ‐tubulin, GCP2, and GCP3, which indicates a role for γTuSCs in the stabilization of its components. [ABSTRACT FROM AUTHOR]

In the N‐degron pathway of protein degradation of Escherichia coli, the N‐recognin ClpS identifies substrates bearing N‐terminal phenylalanine, tyrosine, tryptophan, or leucine and delivers them to the caseinolytic protease (Clp). Chloroplasts contain the Clp system, but whether chloroplastic ClpS1 adheres to the same constraints is unknown. Moreover, the structural underpinnings of substrate recognition are not completely defined. We show that ClpS1 recognizes canonical residues of the E. coli N‐degron pathway. The residue in second position influences recognition (especially in N‐terminal ends starting with leucine). N‐terminal acetylation abrogates recognition. ClpF, a ClpS1‐interacting partner, does not alter its specificity. Substrate binding provokes local remodeling of residues in the substrate‐binding cavity of ClpS1. Our work strongly supports the existence of a chloroplastic N‐degron pathway. [ABSTRACT FROM AUTHOR]

Homeodomain‐interacting protein kinase 2 (HIPK2) is a highly conserved, constitutively active Ser/Thr protein kinase that is involved in various important biological processes. HIPK2 activates itself by auto‐phosphorylation during its synthesis, and its activity is mainly controlled through modulation of its expression by ubiquitin‐dependent degradation. By comparing the expression of wild‐type and kinase‐defective HIPK2, we have recently described a novel mechanism of HIPK2 regulation that is based on preferential co‐translational degradation of kinase‐defective versus wild‐type HIPK2. Here, we have addressed this novel regulatory mechanism in more detail by focusing on the possible involvement of chaperones. Our work shows that HIPK2 is a client of the CDC37‐HSP90 chaperone complex and points to a novel role of CDC37 in the co‐translational degradation of a client protein. [ABSTRACT FROM AUTHOR]

Mitochondria contain more than 1000 different proteins, including several proteolytic enzymes. These mitochondrial proteases form a complex system that performs limited and terminal proteolysis to build the mitochondrial proteome, maintain, and control its functions or degrade mitochondrial proteins and peptides. During protein biogenesis, presequence proteases cleave and degrade mitochondrial targeting signals to obtain mature functional proteins. Processing by proteases also exerts a regulatory role in modulation of mitochondrial functions and quality control enzymes degrade misfolded, aged, or superfluous proteins. Depending on their different functions and substrates, defects in mitochondrial proteases can affect the majority of the mitochondrial proteome or only a single protein. Consequently, mutations in mitochondrial proteases have been linked to several human diseases. This review gives an overview of the components and functions of the mitochondrial proteolytic machinery and highlights the pathological consequences of dysfunctional mitochondrial protein processing and turnover. [ABSTRACT FROM AUTHOR]

The tobacco variant Nicotiana benthamiana has recently emerged as a versatile host for the manufacturing of protein therapeutics, but the fidelity of many recombinant proteins generated in this system is compromised by inadvertent proteolysis. Previous studies have revealed that the anti‐HIV‐1 antibodies 2F5 and PG9 as well as the protease inhibitor α1‐antitrypsin (A1AT) are particularly susceptible to N. benthamiana proteases. Here, we identify two subtilisin‐like serine proteases (NbSBT1 and NbSBT2) whose combined action is sufficient to account for all major cleavage events observed upon expression of 2F5, PG9 and A1AT in N. benthamiana. We propose that downregulation of NbSBT1 and NbSBT2 activities could constitute a powerful means to optimize the performance of this promising platform for the production of biopharmaceuticals. Databases: NbSBT sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers MN534996 to MN535005. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is an important intracellular compartment in eukaryotic cells and has diverse functions, including protein synthesis, protein folding, lipid metabolism and calcium homeostasis. ER functions are disrupted by various intracellular and extracellular stimuli that cause ER stress, including the inhibition of glycosylation, disulphide bond reduction, ER calcium store depletion, impaired protein transport to the Golgi, excessive ER protein synthesis, impairment of ER‐associated protein degradation and mutated ER protein expression. Distinct ER stress signalling pathways, which are known as the unfolded protein response, are deployed to maintain ER homeostasis, and a failure to reverse ER stress triggers cell death. Sphingolipids are lipids that are structurally characterized by long‐chain bases, including sphingosine or dihydrosphingosine (also known as sphinganine). Sphingolipids are bioactive molecules long known to regulate various cellular processes, including cell proliferation, migration, apoptosis and cell–cell interaction. Recent studies have uncovered that specific sphingolipids are involved in ER stress. This review summarizes the roles of sphingolipids in ER stress and human diseases in the context of pathogenic events. [ABSTRACT FROM AUTHOR]

Proteolytic processing is an important post‐translational modification affecting protein activity and stability. In the current study, we investigate the N‐terminal cleavage of Trop2, a protein which is overexpressed in many cancers. We demonstrate that Trop2 is cleaved at Arg87 by a transmembrane serine protease, matriptase. Homology modeling and site‐directed mutagenesis of amino acids in close proximity to the matriptase cleavage site reveal the importance of Val194 in regulating Trop2 cleavage. Co‐immunoprecipitation studies confirm that amino acid substitutions at Arg87, Thr88, Lys189, Val194, and His195 do not affect Trop2 dimerization. However, cleavage of wild‐type Trop2 by matriptase is inhibited when it is allowed to dimerize with a V194A mutant monomer, further confirming the role of Val194 in matriptase‐mediated N‐terminal cleavage. [ABSTRACT FROM AUTHOR]

Autophagy is an intracellular process that regulates the degradation of cytosolic proteins and organelles. Dying cells often accumulate autophagosomes. However, the mechanisms by which necroptotic stimulation induces autophagosomes are not defined. Here, we demonstrate that the activation of necroptosis with TNF‐α plus the cell‐permeable pan‐caspase inhibitor Z‐VAD induces LC3‐II and LC3 puncta, markers of autophagosomes, via the receptor‐interacting protein kinase 3 (RIPK3) in intestinal epithelial cells. Surprisingly, necroptotic stimulation reduces autophagic activity, as evidenced by enlarged puncta of the autophagic substrate SQSTM1/p62 and its increased colocalization with LC3. However, necroptotic stimulation does not induce the lysosomal‐associated membrane protein 1 (LAMP1) nor syntaxin 17, which mediates autophagosome–lysosome fusion, to colocalize with LC3. These data indicate that necroptosis attenuates autophagic flux before the lysosome fusion step. Our findings may provide insights into human diseases involving necroptosis. [ABSTRACT FROM AUTHOR]

Acylaminoacyl peptidases (AAPs) play a pivotal role in various pathological conditions and are recognized as potential therapeutic targets. AAPs exhibit a wide range of activities, such as acylated amino acid-dependent aminopeptidase, endopeptidase, and less studied carboxypeptidase activity. We have determined the crystal structure of an AAP from Geobacillus stearothermophilus (S9gs) at 2.0 Å resolution. Despite being annotated as an aminopeptidase in the NCBI database, our enzymatic characterization proved S9gs to be a carboxypeptidase. Solution-scattering studies showed that S9gs exists as a tetramer in solution, and crystal structure analysis revealed adaptations responsible for the carboxypeptidase activity of S9gs. The findings present a hypothesis for substrate selection, substrate entry, and product exit from the active site, enriching our understanding of this rare carboxypeptidase., (© 2024 Federation of European Biochemical Societies.)

The adenovirus E1B 55K (E1B) protein plays major roles in productive adenoviral infection and cellular transformation. Interest in E1B increased because of the potential of adenoviruses as therapeutic vectors, and the E1B gene is commonly deleted from adenovirus vectors for anticancer therapy. E1B activities are spatiotemporally regulated through SUMOylation and phosphorylation, and through interactions with multiple partners that occur presumably at different intracellular sites and times postinfection. E1B is implicated in the formation of viral replication compartments and regulates viral genome replication and transcription, transcriptional repression, degradation of cellular proteins, and several intranuclear steps of viral late mRNA biogenesis. Here, we review advances in our understanding of E1B during productive adenovirus replication and discuss fundamental aspects that remain unresolved. [ABSTRACT FROM AUTHOR]

Here, we will review the evidence showing that mitotic exit is initiated by regulated proteolysis and then driven by the PPP family of phosphoserine/threonine phosphatases. Rapid APC/CCDC20 and ubiquitin‐dependent proteolysis of cyclin B and securin initiates sister chromatid separation, the first step of mitotic exit. Because proteolysis of Aurora and Polo family kinases dependent on APC/CCDH1 is relatively slow, this creates a new regulatory state, anaphase, different to G2 and M‐phase. We will discuss how the CDK1‐counteracting phosphatases PP1 and PP2A‐B55, together with Aurora and Polo kinases, contribute to the temporal regulation and order of events in the different stages of mitotic exit from anaphase to cytokinesis. For PP2A‐B55, these timing properties are created by the ENSA‐dependent inhibitory pathway and differential recognition of phosphoserine and phosphothreonine. Finally, we will discuss how Aurora B and PP2A‐B56 are needed for the spatial regulation of anaphase spindle formation and how APC/C‐dependent destruction of PLK1 acts as a timer for abscission, the final event of cytokinesis. [ABSTRACT FROM AUTHOR]

CREB3 is a transcription factor localized to the ER. Here, we investigated endogenous CREB3 expression in HEK293 cells using pharmacological and genome editing approaches. Full‐length CREB3 detected under resting conditions disappeared following treatment with tunicamycin, brefeldin A and nigericin. Treatment with cycloheximide and MG132 indicated that endogenous CREB3 is a proteasome substrate. Using cells deficient for the ER‐associated protein degradation (ERAD) factors SEL1L and Herp, we demonstrate that SEL1L, but not Herp, plays a crucial role in the posttranslational regulation of full‐length CREB3 expression. In addition, kifunensine, an α‐mannosidase inhibitor, remarkably increased full‐length CREB3 expression. Our study suggests that endogenous full‐length CREB3 is a novel substrate for ERAD and identifies unique cellular signals distinct from those in canonical ER stress. [ABSTRACT FROM AUTHOR]

Organelles have been studied traditionally as single units, but a novel concept is now emerging: each organelle has distinct functional zones that regulate specific functions. The Golgi apparatus seems to have various zones, including zones for: glycosylphosphatidylinositol‐anchored proteins; proteoglycan, mucin and lipid glycosylation; transport of cholesterol and ceramides; protein degradation (Golgi membrane‐associated degradation); and signalling for apoptosis. The capacity for these specific functions and the size of the corresponding zones appear to be tightly regulated by the Golgi stress response to accommodate cellular demands. For instance, the proteoglycan and mucin zones seem to be separately augmented during the differentiation of chondrocytes and goblet cells, respectively. The mammalian Golgi stress response consists of several response pathways. The TFE3 pathway regulates the general function of the Golgi, such as structural maintenance, N‐glycosylation and vesicular transport, whereas the proteoglycan pathway increases the expression of glycosylation enzymes for proteoglycans. The CREB3 and HSP47 pathways regulate pro‐ and anti‐apoptotic functions, respectively. These observations indicate that the Golgi is a dynamic organelle, the capacity of which is upregulated according to cellular needs. [ABSTRACT FROM AUTHOR]

The tumor protein p53‐inducible nuclear protein 2 (TP53INP2) has been reported to participate in autophagy by interacting with autophagosome‐localized autophagy‐related protein 8 (Atg8) family proteins, including LC3. Here, we uncover a novel function for TP53INP2 in the autophagic degradation of proteins. We identify the ubiquitin‐interacting motif (UIM) of TP53INP2 that mediates its binding to ubiquitin and ubiquitinated proteins. TP53INP2 lacking the UIM is able to displace autophagic adaptor p62 from LC3, which leads to accumulation of ubiquitinated proteins in cells. Furthermore, overexpression of TP53INP2 lacking the UIM sensitizes cells to chloroquine treatment. Our findings indicate that TP53INP2 may act as a novel autophagic adaptor through recruiting ubquitinated substrates to autophagosomes for degradation. [ABSTRACT FROM AUTHOR]

Escherichia coli HtpX is an M48 family zinc metalloproteinase located in the cytoplasmic membrane. Previous studies suggested that it is involved in the quality control of membrane proteins. However, its in vivo proteolytic function has not been characterized in detail, mainly because the physiological substrates have not been identified and no model substrate that allows sensitive detection of the protease activity is available. We constructed a new model substrate of HtpX and established an in vivo semiquantitative and convenient protease activity assay system for HtpX. This system enables detection of differential protease activities of HtpX mutants carrying mutations in conserved regions. This system would also be useful for investigating the functions of HtpX and its homologs in other bacteria. [ABSTRACT FROM AUTHOR]

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by defects of the hydrolase glycosylasparaginase (GA). Previously, we showed that a Canadian AGU mutation disrupts an obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterizations of a model enzyme corresponding to a Finnish AGU allele, the T234I variant. Unlike the Canadian counterpart, the Finnish variant is capable of a slow autoprocessing to generate detectible hydrolyzation activity of the natural substrate of GA. We have determined a 1.6 Å‐resolution structure of the Finnish AGU model and built an enzyme–substrate complex to provide a structural basis for analyzing the negative effects of the point mutation on KM and kcat of the mature enzyme. Enzyme: Glycosylasparaginase or aspartylglucosaminidase, EC3.5.1.26. [ABSTRACT FROM AUTHOR]

Precise regulation of cellular processes is essential for life. Regarding proteins, many regulatory mechanisms were explored over the years, such as posttranslational modifications (e.g., phosphorylation), enzyme activation or inhibition by small molecules, and modulation of protein–protein interactions. Complete removal of a protein via proteolysis as a regulatory mechanism, however, was denied for a long time, mainly due to economical considerations. Scientists could not believe that a protein which is synthesized at the expense of a lot of energy could be destroyed again. Here, we discuss the landmark discoveries and the use of yeast as a eukaryotic model organism that finally paved the way for our current understanding of proteolysis as an essential regulatory principle in the cell. [ABSTRACT FROM AUTHOR]

Each group of the 56 receptor tyrosine kinases (RTK) binds with one or more soluble growth factors and coordinates a vast array of cellular functions. These outcomes are tightly regulated by inducible post‐translational events, such as tyrosine phosphorylation, ubiquitination, ectodomain shedding, and regulated intramembrane proteolysis. Because of the delicate balance required for appropriate RTK function, cells may become pathogenic upon dysregulation of RTKs themselves or their post‐translational covalent modifications. For example, reduced ectodomain shedding and decreased ubiquitination of the cytoplasmic region, both of which enhance growth factor signals, characterize malignant cells. Whereas receptor phosphorylation and ubiquitination are reversible, proteolytic cleavage events are irreversible, and either modification might alter the subcellular localization of RTKs. Herein, we focus on ectodomain shedding by metalloproteinases (including ADAM family proteases), cleavage within the membrane or cytoplasmic regions of RTKs (by gamma‐secretases and caspases, respectively), and complete receptor proteolysis in lysosomes and proteasomes. Roles of irreversible modifications in RTK signaling, pathogenesis, and pharmacology are highlighted. [ABSTRACT FROM AUTHOR]

Although colorectal cancer (CRC) is a prevalent malignancy of the digestive system, the underlying mechanisms of CRC tumorigenesis are still elusive. Arrestin‐related domain‐containing protein‐3 (ARRDC3) has been reported to promote lysosome‐mediated protein degradation. In the present study, we find that the expression of ARRDC3 is downregulated in CRC specimens. Mechanistically, we reveal that ARRDC3 binds and decreases expression of the oncoprotein YAP, the cotranscription factor of the Hippo pathway. The regulation of the Hippo pathway by ARRDC3 is conserved from Drosophila to mammals. Furthermore, we demonstrate that ARRDC3 plays an anti‐oncogenic role in CRC progression by promoting YAP degradation. Finally, we show that ARRDC3 increases the sensitivity of CRC cells toward chemotherapeutic drugs. Taken together, our findings point to ARRDC3 as a potential target for CRC treatment. [ABSTRACT FROM AUTHOR]

The dorsal cell fate determination factor β‐catenin and its antagonist, the ventral cell fate determination factor Xom, are expressed and distributed in a polarized fashion during early vertebrate embryogenesis. Ubiquitin‐mediated proteolysis has been shown to control the abundance of both β‐catenin and Xom. However, the mechanism of ubiquitin‐mediated proteolysis in regulating dorsoventral patterning remains largely unclear. Our current study shows that Xom induces proteolysis of β‐catenin through GSK3‐mediated phosphorylation of Ser33/37 of β‐catenin. Our findings reveal a novel pathway that regulates β‐catenin stability, and suggest, for the first time, a critical function of ubiquitin‐mediated proteolysis in balancing the integration of dorsal–ventral signals and the polarized distribution of β‐catenin and Xom during dorsoventral axis formation. [ABSTRACT FROM AUTHOR]

The pupylation of cellular proteins plays a crucial role in the degradation cascade via the Pup‐Proteasome system (PPS). It is essential for the survival of Mycobacterium smegmatis under nutrient starvation and, as such, the activity of many components of the pathway is tightly regulated. Here, we show that Pup, like ubiquitin, can form polyPup chains primarily through K61 and that this form of Pup inhibits the ATPase‐mediated turnover of pupylated substrates by the 20S proteasome. Similarly, the autopupylation of PafA (the sole Pup ligase found in mycobacteria) inhibits its own enzyme activity; hence, pupylation of PafA may act as a negative feedback mechanism to prevent substrate pupylation under specific cellular conditions. [ABSTRACT FROM AUTHOR]

Ubiquitination plays a pivotal role in most cellular processes and is critical for protein degradation and signalling. E3 ligases are the matchmakers in the ubiquitination cascade, responsible for substrate recognition and modification with specific polyubiquitin chains. Until recently, it was not clear how the catalytic activity of E3s is modulated, but major recent studies on HECT E3 ligases is filling this void. These enzymes appear to be held in a closed, inactive conformation, which is relieved by biochemical manoeuvres unique to each member, thus ensuring exquisite regulation and specificity of the enzymes. The new advances and their significance to the function of HECT E3s are described here, with a particular focus on the Nedd4 family members. [ABSTRACT FROM AUTHOR]

Cellular differentiation, developmental processes, and environmental factors challenge the integrity of the proteome in every eukaryotic cell. The maintenance of protein homeostasis, or proteostasis, involves folding and degradation of damaged proteins, and is essential for cellular function, organismal growth, and viability . Misfolded proteins that cannot be refolded by chaperone machineries are degraded by specialized proteolytic systems. A major degradation pathway regulating cellular proteostasis is the ubiquitin (Ub)/proteasome system ( UPS), which regulates turnover of damaged proteins that accumulate upon stress and during aging. Despite a large number of structurally unrelated substrates, Ub conjugation is remarkably selective. Substrate selectivity is mainly provided by the group of E3 enzymes. Several observations indicate that numerous E3 Ub ligases intimately collaborate with molecular chaperones to maintain the cellular proteome. In this review, we provide an overview of specialized quality control E3 ligases playing a critical role in the degradation of damaged proteins. The process of substrate recognition and turnover, the type of chaperones they team up with, and the potential pathogeneses associated with their malfunction will be further discussed. [ABSTRACT FROM AUTHOR]

Proteostasis, the controlled balance of protein synthesis, folding, assembly, trafficking and degradation, is a paramount necessity for cell homeostasis. Impaired proteostasis is a hallmark of ageing and of many human diseases. Molecular chaperones are essential for proteostasis in eukaryotic cells, and their function has traditionally been linked to protein folding, assembly and disaggregation. More recent findings suggest that chaperones also contribute to key steps in protein degradation. In particular, Hsp70 has an essential role in substrate degradation through the ubiquitin-proteasome system, as well as through different autophagy pathways. Accumulated knowledge suggests that the fate of an Hsp70 substrate is dictated by the combination of partners (cochaperones and other chaperones) that interact with Hsp70 in a given cell context. [ABSTRACT FROM AUTHOR]

The lysosome-associated transporter proton-coupled amino acid transporter 1 (PAT1) promotes nutrient recycling through releasing luminal amino acids into the cytosol. Using HEK293 cells expressing an EGFP-tagged PAT1 ( EGFP- PAT1) as a model, we identified a consensus tyrosine-based targeting signal in the cytosolic N-terminal region of PAT1, which facilitates its expression on the lysosome. Interestingly, this signal can be removed via protein cleavage in an amino acid-sensitive manner. The cleavage is suppressed upon amino acid starvation and is induced by amino acid replenishment. However, amino acid deficiency does not suppress the cleavage of amino acid-binding mutants of EGFP- PAT1. Our data support a mechanism, whereby amino acid binding induces PAT1 cleavage to remove a targeting signal, thus suppressing the expression of PAT1 on the lysosome. [ABSTRACT FROM AUTHOR]

Basal autophagy plays an essential role as a protein quality control system. Although it has been demonstrated that the loss of autophagy results in the accumulation of ubiquitin-positive aggregates and the development of neurodegenerative diseases, the precise autophagy substrate(s) remain unclear. Here, we determined whether ubiquitinated proteins are direct substrates for basal autophagy using a fluorescent ratiometric probe for ubiquitin. We show that the degradation of polyubiquitinated proteins is not dependent on basal autophagy. Although ubiquitin-positive aggregates are observed in autophagy knockout cultured cells, the aggregates consist of soluble and mobile polyubiquitinated proteins, which are trapped by p62 without an increase in the total amount of ubiquitinated proteins. These results suggest that ubiquitinated proteins are not major targets for basal autophagy. [ABSTRACT FROM AUTHOR]

The serum protein histidine-rich glycoprotein ( HRG) has been implicated in tissue injury and tumour growth. Several HRG functions are regulated by the divalent metal Zn2+, including ligand binding and proteolytic processing that releases active HRG fragments. Although HRG can bind divalent metals other than Zn2+, the impact of these divalent metals on the biophysical properties of HRG remains poorly understood. We now show that HRG binds Zn2+, Ni2+, Cu2+ and Co2+ with micromolar affinities, but differing stoichiometries, and regulate the release of specific HRG fragments during proteolysis. Furthermore, HRG binding to Zn2+ promotes HRG dimer formation in a Zn2+-concentration- and pH-dependent manner. Our data highlight the complex divalent metal-dependent regulatory mechanisms that govern HRG function. [ABSTRACT FROM AUTHOR]

The N-end rule is a conserved protein degradation pathway that relates the metabolic stability of a protein to the identity of its N-terminal residue. Proteins bearing a destabilising N-terminal residue (N-degron) are recognised by specialised components of the pathway (N-recognins) and degraded by cellular proteases. In bacteria, the N-recognin ClpS is responsible for the specific recognition of proteins bearing an N-terminal destabilising residue such as leucine, phenylalanine, tyrosine or tryptophan. In this study, we show that the putative apicoplast N-recognin from Plasmodium falciparum ( PfClpS), in contrast to its bacterial homologues, exhibits an expanded substrate specificity that includes recognition of the branched chain amino acid isoleucine. [ABSTRACT FROM AUTHOR]